Therapeutic agent for the treatment of melanomas

ABSTRACT

The present invention provides a therapeutic agent for the treatment of a melanoma or melanomas by injection or intravenously including a mixture of: (a) a water soluble dispersible component; (b) an emulsifier component; and (c) a water insoluble carotenoid component in a suitable carrier medium.

This application is a continuation of application Ser. No. 08/525,665,filed Sep. 22, 1995, now U.S. Pat. No. 5,705,180.

FIELD OF THE INVENTION

The invention relates to a therapeutic agent for the treatment of amelanoma or melanomas and a method for such treatment.

BACKGROUND OF THE INVENTION

Melanomas are caused generally by the exposure of skin to sunlight.Persons of fair complexion have the greatest risk especially those whodevelop moles.

Melanomas originate from a change in normal skin cells, melanocytes,which produce the brown pigment melanin we recognise as tan. Moles andfreckles result from areas of the skin with many melanocytes.

The influence of light on melanocytes is one way by which they can bechanged to grow and divide differently, possibly causing a melanoma. Themelanomas may be malignant, spreading to other parts of the body.Melanomas which do not spread are called benign.

Although melanomas normally form on exposed skin they can start inplaces such as the mouth or bowel.

Melanomas grow in size and need to be surgically removed before theyspread and invade other parts of the body. If the melanomas spread tothe inner organs, removal and treatment is more difficult andchemotherapy or radiotherapy need to be employed.

It has been hypothesised that carotenoids and in particularbeta-carotene may reduce the risk of breast, lung, colon, prostate andcervical cancer, heart disease and stroke and may retard maculardegeneration. In this respect, one hypothesis is that in mammalsbeta-carotene is converted to vitamin A and vitamin A analogues orretinoids (see Moon RC: Comparative aspects of carotenoids and retinoidsas chemopreventive agents for cancer. J Nutr 119:127-134, 1989). It isthis pro-vitamin A activity and the ability to prevent oxidative damagethat has made carotenoids and in particular beta-carotene a compound ofinterest in chemopreventive studies. For instance, anti-oxidants areused, amongst other things, to quench free radicals that are by-productsof normal metabolism in cells.

Beta-carotene has also been used in the treatment of erythropoieticprotoporphyria (EPP). EPP is a genetic disease causing an inadequacy inthe metabolism of porphyrin compounds. It results in a rapid blisteringof the skin on exposure to sunlight.

When considering the use of carotenoid compositions for humanapplication an immediate difficulty arises as a result of the nature ofcarotenoids.

Carotenoids are lipophyllic and therefore not soluble in water in usefulquantities. It is believed that they are transported in the bloodstreamas low density lipoproteins.

The current principal means by which carotenoids are introduced into thebody is orally. However, this method is often unsatisfactory because thepoor absorption of the carotenoid composition by the alimentary canallimits the concentrations in the blood which can be achieved. Further,there will be a substantial delay before a required level of carotenoidsin the bloodstream or a specific organ is reached. Sometimes therequired level cannot be reached as certain individuals do not absorbcarotenoids very well, especially beta-carotene. There is about atenfold difference in the ability of human individuals to absorbbeta-carotene. There have been over 500 carotenoids isolated, but onlyapproximately 15 have been shown to occur in the bloodstream.

Physicians often seek to administer compounds by injection or byintravenous drip rather than oral ingestion. However, because of thevirtual water insolubility of carotenoid compositions it is verydifficult to administer them either by injection or intravenously. Thecompound must be made dispersible in an aqueous base so that it isavailable to the body's cells. In this regard, the base must becompatible with, for example, the bloodstream or lymph, and the materialmust be prepared in a biologically sterile form. The base must itself benon-toxic to the human cells.

To date several in vitro studies have taken place to determine theeffect of beta-carotene on normal and transformed cell types usingsolvents to solubilise the beta-carotene such as tetrahydrofuran,butanol, chloroform, hexane, dimethylsulfoxide, ethanol or in a liposomemicelle. Previous liposome preparations have shown toxicity in cell linecultures as well as being limited in application (see Bertram J S, PungA, Churley M, et al: Diverse carotenoids protect against chemicallyinduced neoplastic transformation. Carcinogenesis 12:671-678, 1991;Hazuka M B, Prasad-Edwards J, Newman F, et al: Beta-carotene inducesmorphological differentiation and decreases adenylate cyclase activityin melanoma cells in culture. J Am Coll Nutr 9:143-149, 1990; Schultz TD, Chew B P, Seaman W R, et al: Inhibitory effect of conjugated dienoicderivatives of linoleic acid and beta-carotene on the in vitro growth ofhuman cancer cells. Canc Letters 63:125-133, 1992; Schwartz J L, ShklarG: The selective cytotoxic effect of carotenoids and a-tocopherol onhuman cancer cell lines in vitro. J Oral Maxillofac Surg 50:367-373,1992; Schwartz J L, Tanaka J, Khandekar V, et al: Beta-Carotene and/orVitamin E as modulators of alkylating agents in SCC-25 human squamouscarcinoma cells. Canc Chemother Pharmacol 29:207-213, 1992; Zhang L-X,Cooney R V, Bertram J S: Carotenoids enhance gap junctionalcommunication and inhibit lipid peroxidation in C3H/10T1/2 cells:relationship to their cancer chemopreventive action. Carcinogenesis12:2109-2114, 1991; and Zhang L-X, Cooney R V, Bertram J S: Carotenoidsup-regulate connexin 43 gene expression independent of their provitaminA or antioxidant properties. Canc Res 52:5707-5712, 1992). Thesesolvents have been found to have a toxic effect which is dose dependent.These solvents are also incompatible with human blood or lymph for thepurposes of intravenous or injectable preparations.

Accordingly, investigations were carried out to develop a carotenoidcomposition which could be accepted by the human body and other animalsand display efficacy in the treatment of melanomas.

DESCRIPTION OF THE INVENTION

In one embodiment of the invention a therapeutic agent is provided forthe treatment of a melanoma or melanomas including a mixture of:

(a) a water soluble or dispersible component;

(b) an emulsifier component, and

(c) a water insoluble carotenoid component in a suitable carrier medium.

Preferably, the water soluble or dispersible component is in the rangeof 30% to 90% by weight.

In a further preferred form of the invention, the water soluble ordispersible component is selected from sugar alcohols, sugars, aminoacids, water, vitamins, blood serum or plasma, lymph, buffers andcombinations and polymers of these materials, and injectables that arewell known in the industry such as mineral salt preparations anddextrose solutions or combinations of these components.

More preferably, the sugar alcohol is glycerol and in yet a furtherpreferred embodiment, glycerol is in the range of 30% to 90% by weight.

In yet a further preferred form of the invention, the emulsifiercomponent is in the range of 0.2% to 20% by weight and more preferably1.0% to 10% by weight.

In yet another further preferred embodiment of the invention, theemulsifier component is selected from glycerides (including preferablymonoglyceride and diglyceride structures from plant and animal sources),polyglycerol esters, lecithins and other phospholipids. More preferablythe glyceride is glyceryl mono-oleate.

In another preferred form of the invention, the water insolublecarotenoid component is beta carotene. In yet a further preferred formthat water insoluble carotenoid component comprises a predominantly 2%to 50% by weight beta-carotene in soya bean oil composition. Morepreferably, the beta carotene concentration is a predominantly 20% to40% by weight and most preferably predominantly 30% by weight.

Preferably, the beta-carotene is a mixture of cis beta-carotene and alltrans beta-carotene. Typically, the cis beta-carotene content of thebeta-carotene is in the range of 50% and 90%, more preferably 70% and85%. More preferably, the beta-carotene is predominantly 9 cisbeta-carotene in a preferred range of 60% to 90%. In an even morepreferred embodiment, the active carotenoid component of the compositionis in the range of 0.1% to 10% by weight and more preferably 1% to 5% byweight.

In yet a further preferred form of the invention, the carrier mediumused to carry the water insoluble carotenoid component is selected fromthe group comprising fatty acids and triglyceride lipids andnon-saponifiable lipid preparations, certain suitable petroleumhydrocarbons including octadecane and combinations of the foregoingcompounds.

In yet a further preferred form of the invention, the triglyceridelipids are selected from the group comprising fats and/or oils derivedfrom plant sources such as seed oils including soya bean, cotton seedand sunflower and from animal sources including fish and beef. Morepreferably, the carrier medium is in the range of 0.1% to 40% by weightand even more preferably 1% to 20%.

In a preferred form of the invention, the agent is diluted for directintroduction into the bloodstream or melanoma or melanomas and morepreferably, the diluting solution is selected from aqueous buffers,normal intravenous preparations (including isotonic saline or 5%dextrose solution) and blood serum and combinations of the foregoing foradministration to cells in vivo and cell line culture media foradministration to cells in vitro.

In a further form of the invention, a method of treatment of a melanomaor melanomas is provided including the step of introducing directly intothe bloodstream or melanoma or melanomas, an effective amount of atherapeutic agent as described above. Preferably, the effective amountis from 0.1 to 10.0 micrograms/ml and more preferably, 0.3 to 3.0)micrograms/ml of the therapeutic agent contacting the melanoma cells.

More preferably, the therapeutic agent is directly introduced into thebloodstream by injection or intravenously. Even more preferably, thetherapeutic agent is injected directly into the site of the melanoma ormelanomas.

The term "mixture" as used herein is intended to include variousphysical forms including emulsions, solutions and crystal suspensions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are graphs showing the effect of carriers on DNA synthesis inhuman metastatic melanoma and neonatal melanocytes. In summary, themelanoma cell strain, c81-46a and melanocytes were incubated for 72hours with soybean oil extract, tetrahydrofuran and a 3:1 mixture ofdimethylsulfoxide:ethanol. Each data point is the mean of 6 wells +/-percent standard error as compared to control.

FIG. 2 is a graph showing the effect of beta-carotene on DNA synthesisin human metastatic melanoma and neonatal melanocytes. In summary,melanoma cell strains: c83-2c, c81-46c, c81-46a, c81-61 and melanocytes(MC) were incubated for 72 hours with beta-carotene. Each data point isthe mean of 6 wells +/- percent standard error as compared to control.Diluent (soy) concentration=0.05%.

FIG. 3 is a graph showing the effect of beta-carotene on theproliferation of human metastatic melanoma and neonatal melanocytes. Insummary, melanoma cell strains: c83-2c, c81-46c, c81-46a and melanocytes(MC) were incubated for 72 hours with beta-carotene and viabilityassessed by trypan blue exclusion. Each data point is the mean of 3wells +/- percent standard error as compared to control. Diluent (soy)concentration=0.05%.

FIG. 4 is a graph showing the effect of beta-carotene on DNA synthesisin human tumor and normal cell lines. In summary, A431, epidermoidcarcinoma, WiDr, colon adenocarcinoma; WI-38. fetal lung fibroblasts andLu-CSF-1, lung adenocarcinoma were incubated for 72 hours withbeta-carotene. Each data point is the mean of 6 wells +/- percentstandard error as compared to control. Diluent (soy)concentration=0.05%.

FIG. 5 is a graph showing the effect of 9-cis Beta Carotene on DNAsynthesis in human metastatic melanoma and neonatal melanocytes. Insummary, the melanoma cell strain c81-46a and melanocytes were incubatedfor 72 hours with 9-cis beta-carotene. Each data point is the mean of 6wells +/- percent standard error as compared to control. Diluent (soy)concentration 0.05%.

FIGS. 1, 2 and 4 refer to "Percent Tritiated Thymidine Incorporation"which is a measure of DNA synthesis activity.

EXAMPLES

The following examples demonstrate the effectiveness of carotenoidcompositions in the treatment of melanomas and the relative non-toxicityof these compositions.

Details of the experiments conducted in relation to the invention are asfollows.

Materials

(a) Cell Cultures

The method used to isolate and culture melanocytes is a combination ofthe procedures developed by Eisinger and Marko (see Eisinger M. Marko O:Selective proliferation of normal human melanocytes in vitro in thepresence of phorbol ester and cholera toxin. Proc Natl Acad Sci79:2018-2022, 1982) and Halaban and Alfano (see Halaban R. Alfano F D:Selective elimination of fibroblasts from cultures of normal humanmelanocytes. In Vitro 20:45-47, 1984).

Briefly, foreskin samples were collected from newborn infants, and themelanocytes isolated and transferred to a T-75 flask. Primary neonatalmelanocytes were cultivated in MCDB 153 medium (Irvine Sci.) asdescribed by Halaban (see Halaban R, Ghosh S, Baird A: bFGF is theputative natural growth factor for normal human melanocytes. In VitroCell Develop Biol 23:47-52, 1987) and modified by Kath (see Kath R,Rodecsk U, Menssen H D et al: Tumor progression in the human melanocyticsystem: Anticancer Res 9:865-872, 1987). Fibroblast contamination wassuppressed by adding geneticin (250 micrograms/ml) to the medium for 2days. Melanoma cell strains (c81-46a, c81-46c, c81-61, c83-2c) werecultured in F-10 (Fisher Sci.) with 5% fetal calf serum, 5% newborn calfserum (Gemini Sci.), penicillin (100 units/ml) and streptomycin (0.1milligrams/ml) (Sigma). The passage number for the melanoma cell strainsused was less than 8, and the melanocytes was less than 5. The melanomacell strains have previously been characterised (see Bregman M D,Meyskens F L: Inhibition of human malignant melanoma colony-formingcells in vitro by prostaglandin Al. Canc Res 43:1642-1645, 1983; ThomsonS P, Meyskens F L: Methods of measurement of self-renewal capacity ofclonogenic cells from biopsies of metastatic human malignant melanoma.Canc Res 42:4606-4613, 1982; and Yohem K H, Slymen D J, Bregman M D, etal: Radiation survival of murine and human melanoma cells utilizing twoassay systems; monolayer and soft agar. Br 3 Canc 57:64-69, 1987). Anumber of other tumour cell lines were also tested namely, A-431 (ahuman epidermoid carcinoma), WiDr (a human colon adenocarcinoma), WI-38(normal human fetal lung fibroblasts) (all obtained from the AmericanType Culture Collection) and Lu-CSF-1 (a human lung adenocarcinoma)(provided by the University of California at Irvine). These four celllines were cultured in DMEM medium (Fisher Sci.). 5% fetal calf serum,5% newborn calf serum, penicillin (100 units/ml) and streptomycin (0.1milligrams/mil).

(b) Chemicals

The beta-carotene was isolated from the alga Dunaliella salina andretresentcd 85-90% of the total carotenoids, with half of the balanceconsisting of oxycarotenoids (lutein and zeaxanthin) and the remaininghalf of alpha-carotene. Gamma-carotene is normally undetectable ascharacterised by high pressure liquid chromatography. The soya bean oilwas isolated from soya beans. A crystalline suspension of beta-caroteneand soya bean oil was created. This resultant phase was then emulsifiedinto the composition described. It was then sterilised by heat orfiltration. Prior to testing on the cell lines each vial of thecomposition was sub-aliquoted into cryogenic vials (Costar) with a freshvial used for each experiment. Throughout all procedures, beta-carotenewas protected from direct light.

Tetrahydrofuran and ethanol (Fisher Sci.) of the highest qualityavailable was used. Dimethylsulfoxide was purchased from Sigma.

The details of the emulsified beta-carotene composition are as follows:

    ______________________________________                     % by weight    ______________________________________    (i)      Beta-carotene 2.4%    and    (ii)     SOY being:             Soya bean oil 6.8%             Glyceryl mono-oleate                           7.2%             Glycerol      66.7%             Water         16.9%    ______________________________________

The above composition can be prepared by the following method. Acrystalline suspension of beta-carotene in soya bean oil is heated andglyceryl mono-oleate is added. This oil phase is dispersed in theglycerol-water phase by high shear mixing followed by homogenisation at60-70° C. Typically, a homogenisation pressure of 8,000 to 10,000 PSI isused, however, this pressure will vary according to the machine that isused. The resulting product is then sterilized by heat processing.Typically, heat processing is effected by autoclaving at 121° C. for 15minutes in a pack for dispensing (3ml glass vial). Optionally, 0.3% ofthe anti-oxidant tocopherols is added to overcome any toxicity that maydevelop over a period of time.

(c) Experimental Conditions

Incorporation of tritiated thyrmidine into DNA was measured in thefollowing manner. Cells were seeded into a 96 well plate (Falcon) andallowed to grow to 50% confluency (24 hours) after which fresh mediumalone, fresh medium with beta-carotene or fresh medium with a carrier(carrier concentration=0.05%) were added and incubated for 72 hours. DNAsynthesis was measured by labelling with methyl-3H!-thymidine (2.5uCi/ml. 20 Ci/mmol Dupont-New England Nuclear) added to the mediumduring the last 15 hours of the treatment period. After incubation,cells were harvested using a PhD cell harvester (Cambridge ResearchInc.). Radioactivity incorporated was determined by liquid scintillationcounting (LS5000TD, Beckman Instruments) with an efficiency of 62.7%.The data is represented as percent tritiated thymidine incorporation ascompared to control. Each data point is the mean of 6 wells +/- percentstandard error.

Cellular proliferation was determined as follows. Cells were seeded into6 well plates (Falcon) and allowed to grow to 50% confluency (24 hours).Fresh medium and the appropriate compound was added and cells thenincubated for 72 hours. After incubation, cells were harvested with0.25% trypsin and washed. Cells were counted on a Coulter Counter(Coulter Instruments) and viability determined by trypan blue exclusion.Each data point is the mean of 3 wells +/- percent standard error.

Results

To determine the effect of a number of carriers for beta-carotene on theactivity of normal melanocytes and a metastatic melanoma cell strain,c81-46a thymidine incorporation was measured. Tetrahydrofuran ("THF"), a3:1 mixture of dimethylsulfoxide/ethanol ("DMSO/ETOH") and the SOY wereincubated with the cells for 72 hours at 0.005%, 0.05% and 0.5%concentration.

As shown in FIG. 1, the SOY did not effect incorporation of thymidine inthe melanoma cells at any concentration of the diluent. THF had only aslight effect on the melanoma cells, while DMSO/ETOH decreasedincorporation by 40% at the highest concentration.

FIG. 2 shows the effect of beta-carotene in the SOY carrier on normalmelanocytes and four metastatic melanoma cell strains. At aconcentration of 0.1 micrograms/ml, beta-carotene had a slightinhibitory effect on the melanoma cell growth. The most sensitive beingc81-61 with a 20% decrease in DNA synthesis. However, the melanomasshowed a differential response to beta-carotene at 1.0 micrograms/ml,ranging from no inhibition to greater than 40%. At the highestbeta-carotene concentration (10 micrograms/ml), normal melanocytes andtwo of the melanomas (c81-61, c81-46c) were more than 95% inhibited.Although, c81-46a and c83-2c remained 20-25% unaffected.

Additionally, viability, as measured by trypan blue exclusion, of normalmelanocytes and the four metastatic melanomas was determined (FIG. 3).Beta-carotene at 1.0 micrograms/ml reduced viability by 20% while at 10micrograms/ml no viable melanocytes were detected. Most striking isbeta-carotene at the highest dose, which resulted in no viablemelanocyte cells and 60% of the melanomas unaffected.

The response of other tumor types was also assessed for their responseto beta-carotene (FIG. 4). The human epidermoid carcinoma cell line.A431, was unaffected by beta-carotene even at 10 micrograms/ml. Thecolon cell line, the normal lung fibroblasts and the lung adenocarcinomacell line were minimally inhibited (10-20%).

FIG. 5 shows the effect of 9-cis beta carotene in the SOY carrier onnormal melonocytes and one metastatic melonoma cell strain. At aconcentration of 1.0 microgram/ml, both the normal melanocyte and thec81-46a were approximately 50% inhibited. At the higher concentration of9-cis beta carotene (10.0 micrograms/ml), the metastatic melonoma cellstrain was 90% inhibited while the melanocyte was slightly lessinhibited than at 1.0 microgram/ml.

Without wishing to be limited to any specific theory, it appears thatthe mixture as illustrated in the above illustrations is a superfineemulsion.

In vitro studies to date have employed various chemical solvents as thecarrier for beta-carotene. It has been found that the effect of thesesolvents alone is cytotoxic. The current testing as set out above hasrevealed that as measured by tritiated thymidine incorporation, the SOYdid not effect the growth of normal human melanocytes or metastaticmelanoma cell strains. However, solvents such as THF and a DMSO/ETOHmixture inhibited DNA synthesis in a concentration dependent manner.

As a novel carrier for beta-carotene, the SOY allowed the effect ofbeta-carotene to be measured without the confounding toxicity of a harshsolvent carrier that interferes with the response.

Melanocytes were selected as a human cell type which is sensitive toinhibition by a range of chemicals. The test results indicated that SOYdid not inhibit incorporation of tritiated thymidine into the melanocyteDNA or the cell viability as determined by trypan blue exclusion.

Melanomas while typically not as sensitive to inhibition by a range ofchemicals as melanocytes, were even more sensitive to even moderatelevels of beta-carotene. This indicated a differential effect formelanomas.

The claims defining the invention are as follows:
 1. A therapeuticcomposition for the treatment of a melanoma or melanomas comprising:(a)a water soluble or dispersible component; (b) an emulsifier component;(c) a water insoluble carotenoid component in a suitable carrier medium.2. A therapeutic composition according to claim 1 wherein the watersoluble or dispersible component is in the range of 30% to 90% byweight.
 3. A therapeutic composition according to claim 1, wherein thewater soluble or dispersible component is selected from the groupconsisting of sugar alcohols, sugars, amino acids, water, vitamins,blood serum, blood plasma, lymph, buffers, mineral salts, andcombinations thereof.
 4. A therapeutic composition according to claim 3wherein the sugar alcohol is glycerol.
 5. A therapeutic compositionaccording to claim 4, wherein the glycerol is in the range of 30% to 90%by weight.
 6. A therapeutic composition according to claim 1, whereinthe emulsifier component is in the range of 0.2% to 20% by weight.
 7. Atherapeutic composition according to claim 1, wherein the emulsifiercomponent is in the range of 1.0% to 10% by weight.
 8. A therapeuticcomposition according to claim 1, wherein the emulsifier component isselected from the group consisting of glycerides, polyglycerol esters,lecithins, and other phospholipids.
 9. A therapeutic compositionaccording to claim 8, wherein the glyceride is glyceryl mono-oleate. 10.A therapeutic composition according to claim 1, wherein the waterinsoluble carotenoid component is predominantly beta-carotene.
 11. Atherapeutic composition according to claim 1, wherein the waterinsoluble carotenoid component in a suitable carrier medium comprises 2%to 50% by weight beta-carotene in a soya bean oil medium.
 12. Atherapeutic composition according to claim 1, wherein the waterinsoluble carotenoid component in a suitable carrier medium comprises20% to 40% by weight beta-carotene in a soya bean oil medium.
 13. Atherapeutic composition according to claim 1, wherein the waterinsoluble carotenoid component in a suitable carrier medium comprises30% by weight beta-carotene in a soya bean oil medium.
 14. A therapeuticcomposition according to claim 10, wherein the beta-carotene comprises amixture of cis beta-carotene and all trans beta-carotene.
 15. Atherapeutic composition according to claim 14, wherein the cisbeta-carotene content of the beta-carotene is in the range of 50% and90%.
 16. A therapeutic composition according to claim 14, wherein thecis beta-carotene content of the beta-carotene is in the range of 70%and 85%.
 17. A therapeutic composition according to claim 14, whereinthe cis beta-carotene content is predominantly 9 cis beta-carotene. 18.A therapeutic composition according to claim 14, wherein the cisbeta-carotene content is in the range of 50% and 90% 9 cisbeta-carotene.
 19. A therapeutic composition according to claim 1,wherein the water insoluble carotenoid component is in the range of 0.1%to 10% by weight of the composition.
 20. A therapeutic compositionaccording to claim 1, wherein the water insoluble carotenoid componentis in the range of 1% to 5% by weight of the composition.
 21. Atherapeutic composition according to claim 1, wherein the carrier mediumused to carry the water insoluble carotenoid component is selected fromthe group consisting of fatty acids, triglyceride lipids,non-saponifiable lipids, petroleum hydrocarbons, and combinationsthereof.
 22. A therapeutic composition according to claim 21, whereinthe triglyceride lipids are selected from the group consisting of fatsand/or oils derived from plant sources and from animal sources.
 23. Atherapeutic composition according to claim 1, wherein the carrier mediumis in the range of 1% to 40% by weight of the composition.
 24. Atherapeutic composition according to claim 1, wherein the carrier mediumis in the range of 1% to 20% by weight of the composition.
 25. Atherapeutic composition according to claim 1, wherein the composition isdiluted for direct introduction into the bloodstream or tissue.
 26. Atherapeutic composition according to claim 25, wherein the agent isdiluted by aqueous buffers, normal intravenous preparations, isotonicsaline, 5% dextrose solution, blood serum or combinations thereof.
 27. Amethod of treatment of a melanoma or melanomas comprising introducingdirectly into the bloodstream or melanoma an effective amount of atherapeutic composition according to claim
 1. 28. The method oftreatment of melanoma or melanomas according to claim 27, wherein theeffective amount is from 0.1 to 10.0 micrograms/ml of the therapeuticcomposition contacting the melanoma cells.
 29. The method of treatmentof melanoma or melanomas according to claim 27, wherein the effectiveamount is from 0.1 to 3.0 micrograms/ml of the therapeutic compositioncontacting the melanoma cells.
 30. A method of treatment of melanoma ormelanomas according to claim 27, wherein the introduction of thetherapeutic composition into the bloodstream is by injection orintravenously.
 31. A method of treatment of a melanoma or melanomasaccording to claim 27, wherein the introduction of the therapeutic agentdirectly into the site of the melanoma or melanomas is by injection. 32.A therapeutic composition for the treatment of a melanoma or melanomas,comprising:(a) a water soluble or dispersible component; (b) anemulsifier component; and (c) a water insoluble carotenoid component ina suitable carrier medium,wherein said water soluble or dispersiblecomponent is in the range of 30% to 90% by weight.
 33. A method oftreatment of melanoma or melanomas comprising the step of introducingdirectly into the bloodstream or melanoma an effective amount of atherapeutic composition according to claim 32.